Fox squirrels (Sciurus niger) (five of eight) were infected with West Nile virus (WNV) when challenged by the oral route with 10^2.3 or 10^3.4 plaque forming units (PFU). The mean maximum serum WNV titer of infected fox squirrels was 10^5.1 PFU/mL and ranged from 10^4.6 to 10^5.6 PFU/mL. These levels of viremia are infectious for several mosquito vectors of WNV. This virus was also isolated from swabs of the oral and rectal cavities, and urine swabs between day 5 and 9 postexposure (p.e.) in amounts as high as 10^2.0, 10^2.8, and 10² PFU, respectively. WNV RNA was detected in salivary gland and/or kidney tissue of three squirrels between day 65 and 72 p.e. in the presence of WNV neutralizing antibody, suggesting that long-term persistent infection occurs in fox squirrels. These observations justify further studies to determine if nonarthropod transmission and long-term persistent infection occur naturally in fox squirrels and contribute to trans-seasonal maintenance of WNV.